mmg_233_2013_genetics_genomicswikiaorg-20200214-history
Lateral transfer of genes involved in nucleotide biosynthesis in C. parvum
Introduction Cryptosprodium parvum is an apicomplexan parasite that is of medical importance. Along with C. hominis it causes the diarrheal disease, cryptosporidiosis. Healthy adults show symptoms upon infection with the parasite but their immune system can eventually cure the infection. Cryptosporidiosis is a major problem in immunocompromised individuals, leading to death in some cases, and in infants below 12 months of age, where cause major growth defects and stunting. There are no drugs or vaccines against the disease in these populations, lending importance to drug development against the pathogen. Enzymes-pathway.jpg|Figure 1: Nucleotide biosynthesis in Apicomplexan parasites Overview.jpg|Figure 2: Predicted model of nucleotide biosynthesis in Cryptosporidium parvum. The enzymes marked in red show strong homology to eubacteria, and those in green show strong phylogenetic association with plants and algae. Strategy C. parvum is an obligate intracellular parasite that replicates in the host, humans. Hence, to treat the pathogen, drugs that are selective to the parasite and not the host have to be developed. The parasite has evolved over time as a product of secondary endosymbiosis from an algae (1). Various studies have been conducted to identify plant and bacterial genes obtained by this eukaryotic parasite by lateral gene transfer (2). The proteins encoded by these genes could be used as targets for drug development. Purine and pyrimidine biosynthesis provides nucleotides important for various vital functions of the parasite including DNA and RNA synthesis, and metabolic pathways. Borris et al. took advantage of both these strategies and mined the genome of C. parvum to identify horizontally transferred genes involved in the nucleotide biosynthesis pathway that could be potential therapeutic targeted (3, 4). Methodology and Results To start with, a genomic analysis of the enzymes involved in nucleotide biosynthetic pathway of different apicomplexan parasites (including Plasmodium falciparum, Toxoplasma gondii, Theileria annulata) was carried out (Figure 1). The sequences of all the apicomplexan parasites were obtained from the Eukaryotic Pathogen database (EuPathDB). Sequences of enzymes from these parasites were used to search for homologous sequences in the other apicomplexan parasites using TBLASTX. The query sequence used for search of each enzyme is shown in the query column of Figure 1. Apicomplexan parasites, in general, do not posses purine synthetic pathway, instead salvage purines from the host. These results show that C. parvum lacks enzymes required for pymidine synthesis as well and can only salvage nucleotides from host cell. Also, the enzymes required for salvage of pyrimidine are not conserved throughout the apicomplexan parasites, and are present only in C. parvum. These enzymes are uridine kinase-uracil phosphoribosyltransferase (UK-UPRT) and thymidine kinase (TK). T. gondii has only the UPRT enzyme but not the fused UK-UPRT enzyme. Furthermore, C. parvum ''has a purine salvage enzyme, inosine 5' monophosphate dehydrogenase (IMPDH) whose sequence matches better with bacterial enzyme rather than apicomplexan parasites. This preliminary data was further validated by conducting phylogenetic analysis to find strong sequence homologs and were also experimentally tested to rule out errors in sequence generation and/or analysis. The authors came up with a model for nucleotide biosynthesis in ''C. parvum ''based on these studies, showing the enzymes obtained from plants/algae and bacteria (Figure 2). UK-UPRT.jpg|Figure 3: Phylogenetic analysis of Crypto UK-UPRT gene TK.jpg|Figure 4: Thymidine Kinase specific to Crypto among Apicoplasts '''Phylogenetic analysis' Multiple sequence alignment was carried out for the genes under study using CLUSTAL X 1.8, and the possible phylogenetic trees constructed. To determine the best fit phylogenetic tree, bootstrap values were obtained using maximum parsimony analysis using paup version 4.0b10 (5), distace analyses using phylip v3.6a3 programs protdist, neighbor, seqboot, and consense and maximun likelihood puzzle frequencies using tree-puzzle version 5.1 for Unix (6). In simpler terms, boostrap values are obtained to check if the mutiple sequence alignment is true or not. For example, there could be a perfect match between sections of sequences due to conserved domain, say ATPase activity, which is in general conserved among several proteins and is not unique to the identify of the select sequences under study. This giving a false homology. To avoid this, bootstrapping is done using different methods, for example, in one method, the complete length of 2 sequences is kept the same, and the columns varied. Small amount of noise may also be introduced in these sequences and homology sampled. Scores for different ways of bootstrapping are obtained. Sequences that show a high homology among by different methods give better confidence to the data. In this study more than 1000 bootstap values were obtained and their percent calculated and shown in the figures of phylogenetic trees. Bootstrap values calculated by maximum parsimony, neighbor-joining and maximum likelihood methods are calculated and shown where possible, in the same order, above the branches in the figures. Uridine kinase-uracil phosphoribosyltransferase (UK-UPRT) The multiple sequence alignment, followed by phylogenetic analysis show that C. parvum's ''UK-UPRT fusion gene is of plant and algae origin (Figure 3). '''Thymidine kinase (TK)' C. parvum ''TK showed strong phylogenetic asssociation to the eubacterial sequences (Figure 4A). Probe of ''C. parvum DNA with TK sequence on a southern blot confirms the presence of this gene in'' C. parvum'' (Figure 4B). Experiments were then conducted using a synthetic analog of thymidine, called bromodeoxyuridine (BrdU) to detect the presence of functional TK in C. parvum (Cp). In these assays, BrdU was added to the host celld before infection of cells with C. parvum. ''Since ''C. parvum ''has a functional TK, it salvaged BrdU from the host cell and used it to synthesize its DNA. Antibodies specific to BrdU (αBrdU) and ''C. parvum (mAbc3c3) showed the presence of BrdU in C. parvum ''fluorescent microscopy (Figure C). DAPI and propidium iodide (PI) are nuclei stains used. When host cells were not treated BrdU, there was no BrdU stain detected in ''C. parvum. Also, T. gondii (Tg) , that does not have the TK enzyme stained negative for BrdU. IMPDH-tree-otherpaper.jpg|Figure 5: IMPDH from Epsilonproteobacteria AK-activity.jpg|Figure 6: Active Adenosine Kinase of Crypto in Toxoplasma gondii Inosine 5' monophosphate dehydrogenase (IMPDH) Figure 5 shows the phylogenetic tree created for IMPDH enzyme. From this tree it is very clear that C. parvum ''IMPDH has a very strong homology to epsilonprotebacteria (Figure 5). '''Adenosine Kinase (AK)' Experiments were carried out to show that AK is the sole source of purine for C. parvum. Since C. parvum cannot be gentically manipulated, these studies were done in T. gondii, as T. gondii also has a functional AK (Figure 1). T. gondii carrying a null mutant of AK (ΔAK) was made and this was complemented by C. parvum AK (ΔAK/CAT-CpAK). Enzyme activity of AK was measured in these T. gondii ''mutants using radiolabeled adenine, which would be incorporated as adenosine monophosphate (AMP) if the enzyme is functional. The radioactivity of the radiolabeled AMP was measured as counts per minute (cpm), which means higher the cpm, higher the enzymatic activity of AK. Figure 6A shows that the wild type ''T. gondii (RH) has a completely functional AK, and complementation of C. parvum AK partially restores the function of this enzyme compared to the null mutant. The same is shown with the help of a monolayer disruption assay and an AK acitvated prodrug called adenosine-arabinoside (AraA). Figure 6B shows that the functional AK activated the prodrug AraA which inhibits T. gondii growth and thus prevents destruction of monolayer (white wells). As a control, the T. gondii null mutant is not affected by the AraA drug. These results suggest that C. parvum has a functional AK as a source of purines. References 1. Abrahamsen MS, Templeton TJ, Enomoto S, Abrahante JE, Zhu G, Lancto CA, et al. Complete genome sequence of the apicomplexan, Cryptosporidium parvum. Science. 2004;304(5669):441-5. Epub 2004/03/27. http://www.ncbi.nlm.nih.gov/pubmed/15044751 PubMed. 2. Huang J, Mullapudi N, Lancto CA, Scott M, Abrahamsen MS, Kissinger JC. Phylogenomic evidence supports past endosymbiosis, intracellular and horizontal gene transfer in Cryptosporidium parvum. Genome biology. 2004;5(11):R88. Epub 2004/11/13. http://www.ncbi.nlm.nih.gov/pubmed/15535864 PubMed. 3. Striepen B, Pruijssers AJ, Huang J, Li C, Gubbels MJ, Umejiego NN, et al. Gene transfer in the evolution of parasite nucleotide biosynthesis. Proceedings of the National Academy of Sciences of the United States of America. 2004;101(9):3154-9. Epub 2004/02/20. http://www.ncbi.nlm.nih.gov/pubmed/14973196 PubMed. 4. Striepen B, White MW, Li C, Guerini MN, Malik SB, Logsdon JM, Jr., et al. Genetic complementation in apicomplexan parasites. Proceedings of the National Academy of Sciences of the United States of America. 2002;99(9):6304-9. Epub 2002/04/18. http://www.ncbi.nlm.nih.gov/pubmed/11959921 PubMed. 5. Swofford, D. L. (2001) paup * *: Phylogenic Analysis Using Parsimony ( *and Other Methods)(Sinauer, Sunderland, MA). http://www.sinauer.com/paup-phylogenetic-analysis-using-parsimony-and-other-methods-4-0-beta.html Link. 6. Schmidt HA, Strimmer K, Vingron M, von Haeseler A. TREE-PUZZLE: maximum likelihood phylogenetic analysis using quartets and parallel computing. Bioinformatics. 2002;18(3):502-4. Epub 2002/04/06. http://www.ncbi.nlm.nih.gov/pubmed/11934758 PubMed.